阴道毛滴虫长寿保障基因Tv-LAG1的克隆和表达

Trichomonas vaginalis LAG1 cDNA Cloning and Fusion Protein Expression

目的克隆原生动物阴道毛滴虫(Trichomonasvaginalis,Tv)长寿保障基因[longevityassurancegene1(LAG1)]的同源基因Tv-LAG1。构建Tv-LAG1原核表达重组体及表达其融合蛋白。方法从阴道毛滴虫cDNA表达文库中筛选LAG1的同源基因,用pET41表达载体与Tv-LAG1cDNA克隆构建原核表达重组体并表达融合蛋白,纯化表达产物用SDS-PAGE法鉴定。用纯化的融合蛋白免疫家兔,获得抗血清,采用Western-blot法对抗体特异性进行分析鉴定。结果成功构建pET41/Tv-LAG1原核表达重组体,并表达出预期大小的重组蛋白。用融合蛋白免疫家兔获得的抗体能特异性识别Tv-Lag1p/GST融合蛋白。结论Tv-Lag1p/GST融合蛋白获得高效表达,并成功纯化,初步实验证明具有一定的免疫原性,为研究Tv-Lag1p在模式生物阴道毛滴虫的细胞定位以及其功能奠定了基础。

Objective To clone and express the LAG1 homolog from a Trichomonas vaginalis cDNA expression library, to express the Tv-Laglp/GST fusion protein, and to prepare the antibody against the fusion protein. Method The recombinant plasmid of pET-41/Tv-LAG1 was constructed, and the expression of the fusion protein was induced by IPTG in Escherichia coli BL21. The fusion protein was then purified with Ni-NTA affinity chromatography, and analyzed by SDS-PAGE. An antiserum against the fusion protein was prepared by immunizing a rabbit and tested by Western-blot. Result The recombinant expression plasmid pET-41/Tv-LAG1 was constructed successfully. The Tv-LAG1/GST fusion protein was expressed in E.coli BL21. Western-blot showed that the antiserum detected a single band. Conclusion The fusion protein of Tv-LAG1 was successfully expressed in E.coli and was antigenic.